Regulation of microRNA homeostasis: Implications in bone fracture healing

microRNA 稳态的调节：对骨折愈合的影响

• 批准号: 10016281
• 负责人: Reyad A Elbarbary
• 金额: $ 39.58万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-11 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016281
• 关键词: Address; Adipocytes; Affect; Biology; Bone Marrow; Bone callus; Cell Differentiation process; Cells; Chemicals; Clinical; Complex; Data; Defect; Development; Diet; Fatty acid glycerol esters; Fluorescent in Situ Hybridization; Fracture; Fracture Healing; Gene Expression; Genes; Grant; Histological Techniques; Homeostasis; Human; Hydrogels; Hyperglycemia; Immature Bone; Immunofluorescence Immunologic; Impaired healing; Impairment; In Vitro; Incidence; Investigation; Knock-out; Knockout Mice; Mammalian Cell; Marrow; Measures; Mediating; Mediator of activation protein; Mesenchymal Differentiation; Mesenchymal Stem Cells; Messenger RNA; MicroRNAs; Micrococcal Nuclease; Minerals; Modeling; Molecular; Mus; Musculoskeletal; Non-Insulin-Dependent Diabetes Mellitus; Obese Mice; Obesity; Osteogenesis; PPAR gamma; Pathologic; Pathology; Pathway interactions; Phase; Phenotype; Play; Population; Process; Proteins; Regulation; Reporting; Resources; Risk Factors; Role; Science; Site; Small Interfering RNA; Stains; Testing; Thinness; Time; Tissue-Specific Gene Expression; Type 2 diabetic; United States National Institutes of Health; Work; bone healing; diabetic patient; experimental study; follow-up; genome-wide; genome-wide analysis; healing; high reward; lipid biosynthesis; miRNA expression profiling; mouse model; multipotent cell; nanoparticle; novel; novel therapeutic intervention; novel therapeutics; nuclease; osteogenic; personalized approach; ranpirnase; repaired; role model; statistics; stem cell differentiation; transcription factor; transcriptome sequencing; transcriptomics

Abstract Impaired or delayed fracture healing is a clinical problem that affects >1.5 million people in the US annually; obesity and associated type 2 diabetes (T2D) are significant and independent risk factors in this context. Nearly 34% of the US population is obese, and the number is projected to climb significantly in the coming decade. Therefore, the incidence of obesity/T2D-associated impaired fracture healing will be a growing concern. Despite these sobering statistics, the molecular basis for delayed healing in obesity/T2D remains unknown and begs investigation. Recently, it has been established that impaired fracture healing in the diet- induced obesity (DIO) mouse model, which is an established model of obesity and hyperglycemia, is accompanied by an increased number of adipocytes within fracture callus. We followed up on these studies to discover that Staphylococcal nuclease and tudor domain-containing 1 (Tudor-SN, abbreviated as TSN) promotes adipogenesis in murine primary bone marrow-derived mesenchymal stem cells (BMSCs), as well as in mouse 3T3-L1 and human HprAD preadipocytes, via degrading particular anti-adipogenic microRNAs (miRNAs), including two key miRNAs that inhibit the expression of peroxisome proliferator-activated receptor gamma (PPARg), the master regulator of adipogenesis. Remarkably, we also found that TSN expression is focally elevated within the callus of DIO mice compared to lean mice, co-localizing with PPARg in the woven- bone lining cells, and occurring at time points immediately preceding the adipocyte bloom. Downregulating callus TSN via local delivery of a chemically modified TSN siRNA inhibited adipogenesis and enhanced mineralized callus formation in DIO mice. According to these preliminary data, we propose the central hypothesis that TSN is a key molecular mediator of the delayed bone healing that occurs in obesity/T2D. To test this hypothesis, we propose to execute two Specific Aims. In the first Specific Aim, we will elucidate the role of TSN as a regulator of BMSCs differentiation and a mediator of adipogenesis that promotes the turnover of anti-adipogenic miRNAs. We will use RNA-seq, miR-seq, and RT-qPCR to study the effect of TSN knockout on the mRNA and miRNA pools in primary BMSCs isolated from wild-type and TSN knockout mice. In the second Specific Aim, we will characterize TSN function in delayed bone healing in DIO mice. We will first compare the expression levels of fracture healing-associated genes at various stages of healing in lean and DIO mice. Comparisons will be performed on the transcriptomic level using RNA-seq, RT-qPCR, and multiplex fluorescence in situ hybridization, and on the protein level using immunofluorescence staining. We will also compare the expression levels of miRNAs using miR-seq and RT-qPCR. Finally, we will study the impact of delivering a TSN siRNA to the fracture callus on different stages of healing in lean and DIO mice. Completion of the proposed experiments will enable us to define the role of TSN and its target miRNAs in the process of fracture repair and implicate TSN in obesity/T2D-associated impaired fracture healing.

抽象的 骨折愈合受损或延迟是一个临床问题，每年影响美国超过 150 万人； 在这种情况下，肥胖和相关的 2 型糖尿病 (T2D) 是重要且独立的危险因素。 近 34% 的美国人口肥胖，预计这一数字在未来还将大幅攀升 十年。因此，肥胖/T2D 相关的骨折愈合受损的发生率将会不断增加 忧虑。尽管有这些发人深省的统计数据，肥胖/T2D 延迟愈合的分子基础仍然存在 未知并请求调查。最近，已经确定饮食会损害骨折愈合—— 诱导性肥胖（DIO）小鼠模型是一种已建立的肥胖和高血糖模型， 伴随着骨折愈伤组织内脂肪细胞数量的增加。我们对这些研究进行了跟进 发现葡萄球菌核酸酶和含有tudor结构域1（Tudor-SN，缩写为TSN） 促进小鼠原代骨髓间充质干细胞 (BMSC) 的脂肪形成，以及 在小鼠 3T3-L1 和人 HprAD 前脂肪细胞中，通过降解特定的抗脂肪生成 microRNA (miRNA)，包括抑制过氧化物酶体增殖物激活受体表达的两个关键 miRNA γ (PPARg)，脂肪生成的主要调节因子。值得注意的是，我们还发现 TSN 表达是 与瘦小鼠相比，DIO 小鼠的愈伤组织内局部升高，与 PPARg 共定位于编织物中。 骨衬细胞，并且发生在脂肪细胞绽放之前的时间点。下调 通过局部递送化学修饰的 TSN siRNA 抑制愈伤组织 TSN 抑制脂肪生成并增强 DIO 小鼠矿化愈伤组织的形成。根据这些初步数据，我们建议中央 假设 TSN 是肥胖/T2D 中发生的骨愈合延迟的关键分子介质。到 为了检验这个假设，我们建议执行两个具体目标。在第一个具体目标中，我们将阐明 TSN 作为 BMSC 分化调节剂和促进周转的脂肪生成调节剂的作用 抗脂肪生成 miRNA。我们将使用RNA-seq、miR-seq和RT-qPCR来研究TSN敲除的效果 对从野生型和 TSN 敲除小鼠中分离的原代 BMSC 中的 mRNA 和 miRNA 池进行分析。在 第二个具体目标，我们将描述 TSN 在 DIO 小鼠骨愈合延迟中的功能。我们首先会 比较瘦人和瘦人在不同愈合阶段骨折愈合相关基因的表达水平 DIO 小鼠。将使用 RNA-seq、RT-qPCR 和多重技术在转录组水平上进行比较 荧光原位杂交，并在蛋白质水平上使用免疫荧光染色。我们还将 使用 miR-seq 和 RT-qPCR 比较 miRNA 的表达水平。最后，我们将研究影响 将 TSN siRNA 递送至瘦小鼠和 DIO 小鼠不同愈合阶段的骨折愈伤组织。完成 所提出的实验将使我们能够定义 TSN 及其目标 miRNA 在这一过程中的作用 骨折修复并表明 TSN 与肥胖/T2D 相关的骨折愈合受损有关。